Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid

dc.contributor

Wichita State University. Department of Chemistry

en_US

dc.contributor.author

Wimalasena, Kandatege

en_US

dc.contributor.author

Wimalasena, D. Shyamali

en_US

dc.date.accessioned

2012-02-06T17:16:59Z

dc.date.available

2012-02-06T17:16:59Z

dc.date.issued

1991-09-02

en_US

dc.identifier

1785690

en_US

dc.identifier

0370535

en_US

dc.identifier.citation

Analytical biochemistry. 1991 Sep 2; 197(2): 353-61.

en_US

dc.identifier.issn

0003-2697

en_US

dc.identifier.uri

http://hdl.handle.net/10057/4375

dc.description

Full text of this article is not available in SOAR.

en_US

dc.description.abstract

Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.

en_US

dc.format.extent

353-61

en_US

dc.language.iso

eng

en_US

dc.publisher

Elsevier

en_US

dc.relation.ispartofseries

Analytical biochemistry

en_US

dc.relation.ispartofseries

Anal. Biochem.

en_US

dc.source

NLM

en_US

dc.subject

Research Support, Non-U.S. Gov't

en_US

dc.subject.lcsh

Ascorbic Acid/chemistry

en_US

dc.subject.mesh

Animals

en_US

dc.subject.mesh

Ascorbic Acid/analogs & derivatives

en_US

dc.subject.mesh

Cattle

en_US

dc.subject.mesh

Chemistry, Clinical

en_US

dc.subject.mesh

Chromaffin Granules/enzymology

en_US

dc.subject.mesh

Dopamine beta-Hydroxylase/analysis

en_US

dc.subject.mesh

Kinetics

en_US

dc.subject.mesh

Oxidation-Reduction

en_US

dc.subject.mesh

Phenylenediamines/chemistry

en_US

dc.subject.mesh

Spectrophotometry/methods

en_US

dc.title

Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid